Methods and compositions for regulation of transgene expression

ABSTRACT

Nucleases and methods of using these nucleases for expressing a transgene from a safe harbor locus in a secretory tissue, and clones and animals derived therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of Ser. No. 13/624,217, filedSep. 21, 2012, now U.S. Pat. No. 9,150,847, and claims the benefit ofU.S. Provisional Application Nos. 61/537,349 filed Sep. 21, 2011; U.S.Provisional Application 61/560,506 filed Nov. 16, 2011; and U.S.Provisional Application 61/670,490 filed Jul. 11, 2012, the disclosuresof which are hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure is in the field of genome editing.

BACKGROUND

Gene therapy holds enormous potential for a new era of humantherapeutics. These methodologies will allow treatment for conditionsthat have not been addressable by standard medical practice. Genetherapy can include the many variations of genome editing techniquessuch as disruption or correction of a gene locus, and insertion of anexpressible transgene that can be controlled either by a specificexogenous promoter fused to the transgene, or by the endogenous promoterfound at the site of insertion into the genome.

Delivery and insertion of the transgene are examples of hurdles thatmust be solved for any real implementation of this technology. Forexample, although a variety of gene delivery methods are potentiallyavailable for therapeutic use, all involve substantial tradeoffs betweensafety, durability and level of expression. Methods that provide thetransgene as an episome (e.g. basic adenovirus, AAV and plasmid-basedsystems) are generally safe and can yield high initial expressionlevels, however, these methods lack robust episome replication, whichmay limit the duration of expression in mitotically active tissues. Incontrast, delivery methods that result in the random integration of thedesired transgene (e.g. integrating lentivirus) provide more durableexpression but, due to the untargeted nature of the random insertion,may provoke unregulated growth in the recipient cells, potentiallyleading to malignancy via activation of oncogenes in the vicinity of therandomly integrated transgene cassette. Moreover, although transgeneintegration avoids replication-driven loss, it does not prevent eventualsilencing of the exogenous promoter fused to the transgene. Over time,such silencing results in reduced transgene expression for the majorityof random insertion events. In addition, integration of a transgenerarely occurs in every target cell, which can make it difficult toachieve a high enough expression level of the transgene of interest toachieve the desired therapeutic effect.

In recent years, a new strategy for transgene integration has beendeveloped that uses cleavage with site-specific nucleases to biasinsertion into a chosen genomic locus (see, e.g., co-owned U.S. Pat. No.7,888,121). This approach offers the prospect of improved transgeneexpression, increased safety and expressional durability, as compared toclassic integration approaches, since it allows exact transgenepositioning for a minimal risk of gene silencing or activation of nearbyoncogenes.

One approach involves the integration of a transgene into its cognatelocus, for example, insertion of a wild type transgene into theendogenous locus to correct a mutant gene. Alternatively, the transgenemay be inserted into a non-cognate locus chosen specifically for itsbeneficial properties. See, e.g., U.S. Patent Publication No.20120128635 relating to targeted insertion of a factor IX (FIX)transgene. Targeting the cognate locus can be useful if one wishes toreplace expression of the endogenous gene with the transgene while stillmaintaining the expressional control exerted by the endogenousregulatory elements. Specific nucleases can be used that cleave withinor near the endogenous locus and the transgene can be integrated at thesite of cleavage through homology directed repair (HDR) or by endcapture during non-homologous end joining (NHEJ). The integrationprocess is determined by the use or non-use of regions of homology inthe transgene donors between the donor and the endogenous locus.

Alternatively, the transgene may be inserted into a specific “safeharbor” location in the genome that may either utilize the promoterfound at that safe harbor locus, or allow the expressional regulation ofthe transgene by an exogenous promoter that is fused to the transgeneprior to insertion. Several such “safe harbor” loci have been described,including the AAVS1 and CCR5 genes in human cells, and Rosa26 in murinecells (see, e.g., co-owned U.S. patent applications Ser. Nos.20080299580; 20080159996 and 201000218264). As described above,nucleases specific for the safe harbor can be utilized such that thetransgene construct is inserted by either HDR- or NHEJ-driven processes.

An especially attractive application of gene therapy involves thetreatment of disorders that are either caused by an insufficiency of asecreted gene product or that are treatable by secretion of atherapeutic protein. Such disorders are potentially addressable viadelivery of a therapeutic transgene to a modest number of cells,provided that each recipient cell expresses a high level of thetherapeutic gene product. In such a scenario, relief from the need forgene delivery to a large number of cells can enable the successfuldevelopment of gene therapies for otherwise intractable indications.Such applications would require permanent, safe, and very high levels oftransgene expression. Thus the development of a safe harbor whichexhibits these properties would provide substantial utility in the fieldof gene therapy.

A considerable number of disorders are either caused by an insufficiencyof a secreted gene product or are treatable by secretion of atherapeutic protein. Clotting disorders, for example, are fairly commongenetic disorders where factors in the clotting cascade are aberrant insome manner, i.e., lack of expression or production of a mutant protein.Most clotting disorders result in hemophilias such as hemophilia A(factor VIII deficiency), hemophilia B (factor IX deficiency), orhemophilia C (factor XI deficiency). Treatment for these disorders isoften related to the severity. For mild hemophilias, treatments caninvolve therapeutics designed to increase expression of theunder-expressed factor, while for more severe hemophilias, therapyinvolves regular infusion of the missing clotting factor (often 2-3times a week) to prevent bleeding episodes. Patients with severehemophilia are often discouraged from participating in many types ofsports and must take extra precautions to avoid everyday injuries.

Alpha-1 antitrypsin (A1AT) deficiency is an autosomal recessive diseasecaused by defective production of alpha 1-antitrypsin which leads toinadequate A1AT levels in the blood and lungs. It can be associated withthe development of chronic obstructive pulmonary disease (COPD) andliver disorders. Currently, treatment of the diseases associated withthis deficiency can involve infusion of exogenous A1AT and lung or livertransplant.

Lysosomal storage diseases (LSDs) are a group of rare metabolicmonogenic diseases characterized by the lack of functional individuallysosomal proteins normally involved in the breakdown of waste lipids,glycoproteins and mucopolysaccharides. These diseases are characterizedby a buildup of these compounds in the cell since it is unable toprocess them for recycling due to the mis-functioning of a specificenzyme. Common examples include Gaucher's (glucocerebrosidasedeficiency-gene name: GBA), Fabry's (α galactosidase deficiency—GLA),Hunter's (iduronate-2-sulfatase deficiency-IDS), Hurler's (alpha-Liduronidase deficiency—IDUA), and Niemann-Pick's (sphingomyelinphosphodiesterase 1deficiency—SMPD1) diseases. When grouped together,LSDs have an incidence in the population of about 1 in 7000 births.These diseases have devastating effects on those afflicted with them.They are usually first diagnosed in babies who may have characteristicfacial and body growth patterns and may have moderate to severe mentalretardation. Treatment options include enzyme replacement therapy (ERT)where the missing enzyme is given to the patient, usually throughintravenous injection in large doses. Such treatment is only to treatthe symptoms and is not curative, thus the patient must be givenrepeated dosing of these proteins for the rest of their lives, andpotentially may develop neutralizing antibodies to the injected protein.Often these proteins have a short serum half-life, and so the patientmust also endure frequent infusions of the protein. For example,Gaucher's disease patients receiving the Cerezyme® product(imiglucerase) must have infusions three times per week. Production andpurification of the enzymes is also problematic, and so the treatmentsare very costly (>$100,000 per year per patient).

Type I diabetes is a disorder in which immune-mediated destruction ofpancreatic beta cells results in a profound deficiency of insulin, whichis the primary secreted product of these cells. Restoration of baselineinsulin levels provide substantial relief from many of the more seriouscomplications of this disorder which can include “macrovascular”complications involving the large vessels: ischemic heart disease(angina and myocardial infarction), stroke and peripheral vasculardisease, as well as “microvascular” complications from damage to thesmall blood vessels. Microvascular complications may include diabeticretinopathy, which affects blood vessel formation in the retina of theeye, and can lead to visual symptoms, reduced vision, and potentiallyblindness, and diabetic nephropathy, which may involve scarring changesin the kidney tissue, loss of small or progressively larger amounts ofprotein in the urine, and eventually chronic kidney disease requiringdialysis. Diabetic neuropathy can cause numbness, tingling and pain inthe feet and, together with vascular disease in the legs, contributes tothe risk of diabetes-related foot problems (such as diabetic footulcers) that can be difficult to treat and occasionally requireamputation as a result of associated infections.

Antibodies are secreted protein products whose binding plasticity hasbeen exploited for development of a diverse range of therapies.Therapeutic antibodies can be used for neutralization of target proteinsthat directly cause disease (e.g. VEGF in macular degeneration) as wellas highly selective killing of cells whose persistence and replicationendanger the hose (e.g. cancer cells, as well as certain immune cells inautoimmune diseases). In such applications, therapeutic antibodies takeadvantage of the body's normal response to its own antibodies to achieveselective killing, neutralization, or clearance of target proteins orcells bearing the antibody's target antigen. Thus antibody therapy hasbeen widely applied to many human conditions including oncology,rheumatology, transplant, and ocular disease Examples of antibodytherapeutics include Lucentis® (Genentech) for the treatment of maculardegeneration, Rituxan® (Biogen Idec) for the treatment of Non-Hodgkinlymphoma, and Herceptin® (Genentech) for the treatment of breast cancer.Albumin is a protein that is produced in the liver and secreted into theblood. In humans, serum albumin comprises 60% of the protein found inblood, and its function seems to be to regulate blood volume byregulating the colloid osmotic pressure. It also serves as a carrier formolecules with low solubility, for example lipid soluble hormones, bilesalts, free fatty acids, calcium and transferrin. In addition, serumalbumin carries therapeutics, including warfarin, phenobutazone,clofibrate and phenytoin. In humans, the albumin locus is highlyexpressed, resulting in the production of approximately 15 g of albuminprotein each day. Albumin has no autocrine function, and there does notappear to be any phenotype associated with monoallelic knockouts andonly mild phenotypic observations are found for biallelic knockouts (seeWatkins et at (1994) Proc Natl Acad Sci USA 91:9417).

Albumin has also been used when coupled to therapeutic reagents toincrease the serum half-life of the therapeutic. For example, Osborn etat (J Pharm Exp Thera (2002) 303(2):540) disclose the pharmacokineticsof a serum albumin-interferon alpha fusion protein and demonstrate thatthe fusion protein had an approximate 140-fold slower clearance suchthat the half-life of the fusion was 18-fold longer than for theinterferon alpha protein alone. Other examples of therapeutic proteinsrecently under development that are albumin fusions include Albulin-G™,Cardeva™ and Albugranin™ (Teva Pharmaceutical Industries, fused toInsulin, b-type natriuretic, or GCSF, respectively), Syncria®(GlaxoSmithKline, fused to Glucagon-like peptide-1) and Albuferon α-2B,fused to IFN-alpha (see Current Opinion in Drug Discovery andDevelopment, (2009), vol 12, No. 2. p. 288). In these cases, Albulin-G™,Cardeva™ and Syncria® are all fusion proteins where the albumin is foundon the N-terminus of the fusion, while Albugranin™ and Albuferon alpha2G are fusions where the albumin is on the C-terminus of the fusion.

Thus, there remains a need for additional methods and compositions thatcan be used to express a desired transgene at a therapeutically relevantlevel, while avoiding any associated toxicity, and which may limitexpression of the transgene to the desired tissue type, for example totreat genetic diseases such as hemophilias, diabetes, lysosomal storagediseases and A1AT deficiency. Additionally, there remains a need foradditional methods and compositions to express a desired transgene at atherapeutically relevant level for the treatment of other diseases suchas cancers.

SUMMARY

Disclosed herein are methods and compositions for creating a safe harborin the genome of cells, for targeted insertion and subsequenceexpression of a transgene, for example expression of the transgene froma secretory tissue such as liver. In one aspect, described herein is anon-naturally occurring zinc-finger protein (ZFP) that binds to targetsite in a region of interest (e.g., an albumin gene) in a genome,wherein the ZFP comprises one or more engineered zinc-finger bindingdomains. In one embodiment, the ZFP is a zinc-finger nuclease (ZFN) thatcleaves a target genomic region of interest, wherein the ZFN comprisesone or more engineered zinc-finger binding domains and a nucleasecleavage domain or cleavage half-domain. Cleavage domains and cleavagehalf domains can be obtained, for example, from various restrictionendonucleases and/or homing endonucleases. In one embodiment, thecleavage half-domains are derived from a Type IIS restrictionendonuclease (e.g., Fok I). In certain embodiments, the zinc fingerdomain recognizes a target site in an albumin gene, for example a zincfinger protein with the recognition helix domains ordered as shown in asingle row of Tables 1, 3, 5 or 8.

In another aspect, described herein is a Transcription Activator LikeEffector (TALE) protein that binds to target site in a region ofinterest (e.g., an albumin gene) in a genome, wherein the TALE comprisesone or more engineered TALE binding domains. In one embodiment, the TALEis a nuclease (TALEN) that cleaves a target genomic region of interest,wherein the TALEN comprises one or more engineered TALE DNA bindingdomains and a nuclease cleavage domain or cleavage half-domain. Cleavagedomains and cleavage half domains can be obtained, for example, fromvarious restriction endonucleases and/or homing endonucleases. In oneembodiment, the cleavage half-domains are derived from a Type IISrestriction endonuclease (e.g., Fok I). In certain embodiments, the TALEDNA binding domain recognizes a target site in an albumin gene, forexample TALE DNA binding domain having the target sequence shown in asingle row of Table 12.

The ZFN and/or TALEN as described herein may bind to and/or cleave theregion of interest in a coding or non-coding region within or adjacentto the gene, such as, for example, a leader sequence, trailer sequenceor intron, or within a non-transcribed region, either upstream ordownstream of the coding region. In certain embodiments, the ZFN bindsto and/or cleaves an albumin gene. In other embodiments, the ZFN and/orTALEN binds to and/or cleaves a safe-harbor gene, for example a CCR5gene, a PPP1R12C (also known as AAVS1) gene or a Rosa gene. See, e.g.,U.S. Patent Publication Nos. 20080299580; 20080159996 and 201000218264.In another aspect, described herein are compositions comprising one ormore of the zinc-finger and/or TALE nucleases described herein. Incertain embodiments, the composition comprises one or more zinc-fingerand/or TALE nucleases in combination with a pharmaceutically acceptableexcipient.

In another aspect, described herein is a polynucleotide encoding one ormore ZFNs and/or TALENs described herein. The polynucleotide may be, forexample, mRNA. In some aspects, the mRNA may be chemically modified (Seee.g. Kormann et al, (2011) Nature Biotechnology 29(2):154-157).

In another aspect, described herein is a ZFN and/or TALEN expressionvector comprising a polynucleotide, encoding one or more ZFNs and/orTALENs described herein, operably linked to a promoter. In oneembodiment, the expression vector is a viral vector. In one aspect, theviral vector exhibits tissue specific tropism.

In another aspect, described herein is a host cell comprising one ormore ZFN and/or TALEN expression vectors. The host cell may be stablytransformed or transiently transfected or a combination thereof with oneor more ZFP or TALEN expression vectors. In one embodiment, the hostcell is an embryonic stem cell. In other embodiments, the one or moreZFP and/or TALEN expression vectors express one or more ZFNs and/orTALENs in the host cell. In another embodiment, the host cell mayfurther comprise an exogenous polynucleotide donor sequence.Non-limiting examples of suitable host cells include eukaryotic cells orcell lines such as secretory cells (e.g., liver cells, mucosal cells,salivary gland cells, pituitary cells, etc.), blood cells (red bloodcells), stem cells, etc. In any of the embodiments described herein thehost cell can comprise an embryo cell, for example, of a mouse, rat,rabbit or other mammal cell embryo.

In another aspect, described herein is a method for cleaving an albumingene in a cell, the method comprising: introducing, into the cell, oneor more polynucleotides encoding one or more ZFNs and/or TALENs thatbind to a target site in the one or more albumin genes under conditionssuch that the ZFN(s) is (are) or TALENs is (are) expressed and the oneor more albumin genes are cleaved.

In other embodiments, a genomic sequence in any target gene is replaced,for example using a ZFN or TALEN (or vector encoding said ZFN or TALEN)as described herein and a “donor” sequence (e.g., transgene) that isinserted into the gene following targeted cleavage with the ZFN and/orTALEN. The donor sequence may be present in the ZFN or TALEN vector,present in a separate vector (e.g., Ad or LV vector) or, alternatively,may be introduced into the cell using a different nucleic acid deliverymechanism. Such insertion of a donor nucleotide sequence into the targetlocus (e.g., albumin gene, other safe-harbor gene, etc.) results in theexpression of the transgene carried by the donor under control of thetarget locus's (e.g. albumin) genetic control elements. In some aspects,insertion of the transgene of interest, for example into an albumin generesults in expression of an intact exogenous protein sequence and lacksany albumin encoded amino acids. In other aspects, the expressedexogenous protein is a fusion protein and comprises amino acids encodedby the transgene and by an albumin gene (e.g., from the endogenoustarget locus or, alternatively from albumin-encoding sequences on thetransgene). In some instances, the albumin sequences will be present onthe amino (N)-terminal portion of the exogenous protein, while inothers, the albumin sequences will be present on the carboxy(C)-terminal portion of the exogenous protein. In other instances,albumin sequences will be present on both the N- and C-terminal portionsof the exogenous protein. The albumin sequences may include full-lengthwild-type or mutant albumin sequences or, alternatively, may includepartial albumin amino acid sequences. In certain embodiments, thealbumin sequences (full-length or partial) serve to increase the serumhalf-life of the polypeptide expressed by the transgene to which it isfused and/or as a carrier. In some embodiments, the albumin-transgenefusion is located at the endogenous locus within the cell while in otherembodiments, the albumin-transgene coding sequence is inserted into asafe harbor within a genome. In some aspects, the safe harbor isselected from the AAVS1, Rosa, HPRT or CCR5 locus (see co-owned USpatent publications Nos. 20080299580; 20080159996 and 201000218264, andU.S. Provisional patent application No. 61/556,691).

In another aspect, the invention describes methods and compositions thatcan be used to express a transgene under the control of an albuminpromoter in vivo (e.g., endogenous or exogenous albumin promoter). Insome aspects, the transgene may encode a therapeutic protein ofinterest. The transgene may encode a protein such that the methods ofthe invention can be used for production of protein that is deficient orlacking (e.g., “protein replacement”). In some instances, the proteinmay be involved treatment for a lysosomal storage disease. Othertherapeutic proteins may be expressed, including protein therapeuticsfor conditions as diverse as epidermolysis bullosa or AAT deficientemphysema. In other aspects, the transgene may comprise sequences (e.g.,engineered sequences) such that the expressed protein hascharacteristics which give it novel and desirable features (increasedhalf-life, changed plasma clearance characteristics etc.). Engineeredsequences can also include amino acids derived from the albuminsequence. In some aspects, the transgenes encode therapeutic proteins,therapeutic hormones, plasma proteins, antibodies and the like. In someaspects, the transgenes may encode proteins involved in blood disorderssuch as clotting disorders. In some aspects, the transgenes encodestructural nucleic acids (shRNAs, miRNAs and the like).

In some embodiments, the methods of the invention may be used in vivo intransgenic animal systems. In some aspects, the transgenic animal may beused in model development where the transgene encodes a human gene. Insome instances, the transgenic animal may be knocked out at thecorresponding endogenous locus, allowing the development of an in vivosystem where the human protein may be studied in isolation. Suchtransgenic models may be used for screening purposes to identify smallmolecule, large biomolecules or other entities which may interact ormodify the human protein of interest. In other aspects, the transgenicanimals may be used for production purposes, for example, to produceantibodies or other biomolecules of interest. In certain embodiments,the animal is a small mammal, for example a dog, rabbit or a rodent suchas rat, a mouse or a guinea pig. In other embodiments, the animal is anon-human primate. In yet further embodiments, the animal is a farmanimal such as a cow, goat or pig. In some aspects, the transgene isintegrated into the selected locus (e.g., albumin or safe-harbor) into astem cell (e.g., an embryonic stem cell, an induced pluripotent stemcell, a hepatic stem cell, etc.) or animal embryo obtained by any of themethods described herein, and then the embryo is implanted such that alive animal is born. The animal is then raised to sexual maturity andallowed to produce offspring wherein at least some of the offspringcomprise the integrated transgene.

In a still further aspect, provided herein is a method for site specificintegration of a nucleic acid sequence into an endogenous locus (e.g.,albumin gene) of a chromosome, for example into the chromosome of anembryo. In certain embodiments, the method comprises: (a) injecting anembryo with (i) at least one DNA vector, wherein the DNA vectorcomprises an upstream sequence and a downstream sequence flanking thenucleic acid sequence to be integrated, and (ii) at least one RNAmolecule encoding a zinc finger and/or TALE nuclease that recognizes thesite of integration in the target locus (e.g., albumin locus), and (b)culturing the embryo to allow expression of the zinc finger and/or TALEnuclease, wherein a double stranded break introduced into the site ofintegration by the zinc finger nuclease or TALEN is repaired, viahomologous recombination with the DNA vector, so as to integrate thenucleic acid sequence into the chromosome.

Suitable embryos may be derived from several different vertebratespecies, including mammalian, bird, reptile, amphibian, and fishspecies. Generally speaking, a suitable embryo is an embryo that may becollected, injected, and cultured to allow the expression of a zincfinger or TALE nuclease. In some embodiments, suitable embryos mayinclude embryos from small mammals (e.g., rodents, rabbits, etc.),companion animals, livestock, and primates. Non-limiting examples ofrodents may include mice, rats, hamsters, gerbils, and guinea pigs.Non-limiting examples of companion animals may include cats, dogs,rabbits, hedgehogs, and ferrets. Non-limiting examples of livestock mayinclude horses, goats, sheep, swine, llamas, alpacas, and cattle.Non-limiting examples of primates may include capuchin monkeys,chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys,squirrel monkeys, and vervet monkeys. In other embodiments, suitableembryos may include embryos from fish, reptiles, amphibians, or birds.Alternatively, suitable embryos may be insect embryos, for instance, aDrosophila embryo or a mosquito embryo.

In any of the methods or compositions described herein, the cellcontaining the engineered locus (e.g., albumin locus) can be a stemcell. Specific stem cell types that may be used with the methods andcompositions of the invention include embryonic stem cells (ESC),induced pluripotent stem cells (iPSC) and hepatic or liver stem cells.The iPSCs can be derived from patient samples and from normal controlswherein the patient derived iPSC can be mutated to normal gene sequenceat the gene of interest, or normal cells can be altered to the knowndisease allele at the gene of interest. Similarly, the hepatic stemcells can be isolated from a patient. These cells are then engineered toexpress the transgene of interest, expanded and then reintroduced intothe patient.

In any of the methods described herein, the polynucleotide encoding thezinc finger nuclease(s) and/or TALEN(s) can comprise DNA, RNA orcombinations thereof. In certain embodiments, the polynucleotidecomprises a plasmid. In other embodiments, the polynucleotide encodingthe nuclease comprises mRNA.

Also provided is an embryo comprising at least one DNA vector, whereinthe DNA vector comprises an upstream sequence and a downstream sequenceflanking the nucleic acid sequence to be integrated, and at least oneRNA molecule encoding a zinc finger nuclease that recognizes thechromosomal site of integration. Organisms derived from any of theembryos as described herein are also provided (e.g., embryos that areallowed to develop to sexual maturity and produce progeny).

In another aspect provided by the methods and compositions of theinvention is the use of cells, cell lines and animals (e.g., transgenicanimals) in the screening of drug libraries and/or other therapeuticcompositions (i.e., antibodies, structural RNAs, etc.) for use intreatment of an animal afflicted with a clotting factor disorder. Suchscreens can begin at the cellular level with manipulated cell lines orprimary cells, and can progress up to the level of treatment of a wholeanimal (e.g., human).

A kit, comprising the ZFPs and/or TALENs of the invention, is alsoprovided. The kit may comprise nucleic acids encoding the ZFPs orTALENs, (e.g. RNA molecules or ZFP or TALEN encoding genes contained ina suitable expression vector), donor molecules, suitable host celllines, instructions for performing the methods of the invention, and thelike.

Thus, the disclosure herein includes, but is not limited to, thefollowing embodiments:

1. A non-naturally occurring fusion protein comprising a DNA-bindingprotein that binds to an endogenous albumin gene and a cleavage domain,wherein the fusion protein modifies the endogenous albumin gene.

2. The fusion protein of embodiment 1, wherein the DNA-binding proteincomprises a zinc finger protein.

3. The fusion protein of embodiment 2, wherein the zinc finger proteincomprises 4, 5 or 6 zinc finger domains comprising a recognition helixregion, wherein the zinc finger proteins comprise the recognition helixregions shown in a single row of Table 1, Table 3, Table 5 or Table 8.

4. The fusion protein of embodiment 1, wherein the DNA-binding proteincomprises a TALE DNA-binding domain.

5. The fusion protein of embodiment 4, wherein the TALE DNA-bindingdomain binds to a target sequence shown in a single row of Table 12.

6. A polynucleotide encoding one or more fusion proteins of embodiments1 to 5.

7. An isolated cell comprising one or more fusion proteins according toembodiments 1 to 5 or one or more polynucleotides according toembodiment 6.

8. The cell of embodiment 7, wherein the cell is a stem cell or anembryo cell.

9. The cell of embodiment 8, wherein the stem cell is selected from thegroup consisting of an embryonic stem cell (ESC), an induced pluripotentstem cell (iPSC), a hepatic stem cell and a liver stem cell.

10. A kit comprising a fusion protein according to embodiments 1 to 5 ora polynucleotide according to embodiment 6 or a cell according toembodiment 7-9.

11. A method of cleaving an endogenous albumin gene in a cell, themethod comprising:

introducing, into the cell, one or more expression vectors comprising atleast one polynucleotide according to embodiment 6, under conditionssuch that the one or more fusion proteins are expressed and the albumingene is cleaved.

12. The method of embodiment 11, wherein the polynucleotide comprises anAAV vector.

13. The method of embodiment 11, wherein the cell is a liver cell.

14. A method of introducing a transgene into an endogenous albumin gene,the method comprising:

cleaving the endogenous albumin gene according to the method of any ofembodiments 15-17 in the presence of an exogenous polynucleotidecomprising the transgene such that the transgene is integrated into theendogenous albumin gene.

15. The method of embodiment 14, wherein the transgene expresses atherapeutic protein.

16. The method of embodiment 15, wherein the therapeutic protein isinvolved in treating a lysosomal storage disease, epidermolysis bullosa,AAT deficient emphysema or blood disorders such as clotting disorders.

17. The method of embodiments 15 or 16, wherein expression of thetransgene is driven by the endogenous albumin control sequences.

18. The method of any of embodiments 15-17, wherein the transgenefurther comprises albumin sequences.

19. The method of embodiment 18, wherein the albumin sequences arepresent on the amino (N)-terminal and/or carboxy (C)-terminal portion ofthe protein.

20. A method of increasing the serum half-life of a polypeptideexpressed from a transgene integrated into an endogenous albumin gene,the method comprising introducing the transgene into the endogenousalbumin gene according to the method of embodiment 18 or embodiment 19,wherein the transgene expresses the polypeptide and albumin sequencessuch that the serum half-life of the polypeptide in increased.

21. A method of treating a subject having a disease caused by adeficiency of a polypeptide, the method comprising, introducing atransgene encoding the polypeptide into an isolated cell according tothe method of embodiments 14-19 such that the transgene is expressed inthe isolated cell; and introducing the isolated cell into the subject,thereby treating the disease.

22. The method of embodiment 21, wherein the cell is a liver cell or astem cell.

23. The cell of embodiment 22, wherein the stem cell is selected fromthe group consisting of an embryonic stem cell (ESC), an inducedpluripotent stem cell (iPSC), a hepatic stem cell and a liver stem cell.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are gels depicting the results of a Cel-I mismatch assay(Surveyor™, Transgenomic) that quantifies the degree to which ZFNcleavage of an endogenous chromosomal target, followed by imperfectrepair via NHEJ, has yielded small insertions or deletions (“indels”) ofthe targeted locus. For a description of the assay see Horton et al.Methods Mol Biol. (2010) 649:247-56. FIG. 1A shows results usingexpression constructs for ZFNs targeted to the mouse albumin gene whichwere transfected into Neuro2A cells, where the cells were treated for 3days at 37° C. following transfection, and then analyzed for thefraction of modified target sites via Cel-I analysis. FIG. 1B showsresults for the same ZFNs and cells as FIG. 1A except cells weresubjected to hypothermic shock (30° C.) during their 3 days of growthfollowing transfection. The percent mismatch, or % indels shown at thebottom of the lanes, is a measure of the ZFN activity and demonstratesthat the mouse albumin specific ZFNs are able to induce up to 53% indelsfollowing cleavage of their endogenous chromosomal target in Neuro 2Acells.

FIGS. 2A and 2B are gels depicting the results of a Cel-I mismatch assaycarried out on canine D17 cells transfected with constructs expressingthe canine albumin specific ZFN pair SBS 33115/SBS34077 at twoconcentrations of plasmid DNA, 20 or 40 ng. FIG. 2A depicts the resultsafter 3 days while FIG. 2B depicts the results after 10 days. This ZFNpair was able to induce indels in ˜25-30% of target site sequences atday 3.

FIGS. 3A and 3B show alignments of the albumin genes from a variety ofspecies of interest. FIG. 3A shows an alignment of exon 1 and the 5′region of intron 1 of human (H. sapiens, SEQ ID NO:160), rhesus macaquemonkeys (M. mulatta, SEQ ID NO:73), marmoset (C. jacchus, SEQ ID NO:74),dog (C. familiaris, SEQ ID NO:165), rat (R. norvegicus, SEQ ID NO:165)and mouse (M. musculus, SEQ ID NO:76). 3B shows an alignment of theremainder of intron 1 and a small fragment of exon 2. This regionincludes the Locus 1 to Locus 5 of human (SEQ ID NO:161), rhesus macaquemonkeys (SEQ ID NO:77), marmoset (SEQ ID NO:78), dog (SEQ ID NO:79), rat(SEQ ID NO:80) and mouse (M. musculus, SEQ ID NO:81) which are loci inthe albumin gene chosen for ZFN targeting. The sequences depicted showthe starting codon ATG (large box in FIG. 3A) and the boundaries ofexon1 and intron 1 (FIG. 3A) and intron 1 and exon 2 (FIG. 3B).

FIGS. 4A and 4B depict the results of a Cel-I mismatch assay carried outon genomic DNA from liver tissue biopsied from mice injected withalbumin-specific ZFNs expressed from a hepatotrophic AAV8 vector. Theresults are from 10 wild type mice (numbers 273-282) injectedintravenously via tail vein injection with two sets of ZFN pairs (pair1: SBS30724 and SBS30725 and pair 2: SBS30872 and SBS30873). FIG. 4A isa gel that quantifies the indels present in the amplicon encompassingthe pair 1 site and FIG. 4B is another gel that quantifies the indelspresent in the amplicon that encompasses the pair 2 site. The percent ofalbumin genes bearing ZFN-induced modifications in the liver biopsies isindicated at the bottom of the lanes, and demonstrates that the albuminZFN pairs are capable of modifying up to 17% of targets when thenucleases are delivered in vivo.

FIGS. 5A and 5B show the results of a Cel-I mismatch assay carried outon genomic DNA from liver tissue biopsied from mice injected withalbumin-specific ZFNs expressed from different chimeric AAV vectors.Experimental details are provided in Example 5. FIG. 5A demonstratesthat the ZFNs are able to cleave the albumin target in the liver in vivowhen introduced into the animal via AAV-mediated gene delivery. Thepercent of albumin genes bearing ZFN-induced modifications in the liverbiopsies ranged up to 16 percent. FIG. 5B shows a Western blot of livertissue using either anti-Flag antibodies or anti-p65. The open readingframes encoding the ZFNs were fused to a sequence encoding a polypeptideFLAG-tag. Thus, the anti-Flag antibody detected the ZFNs anddemonstrated ZFN expression in the mice receiving ZFNs. The anti-p65antibody served as a loading control in these experiments and indicatedthat comparable amounts of protein were loaded in each lane.

FIG. 6 shows results from a mouse study in which groups of mice weretreated with the mouse albumin specific ZFN pair 30724/30725 viadelivery of differing doses of different AAV serotypes, and thenassessed for gene modification using the Cel- I assay. The AAV serotypestested in this study were AAV2/5, AAV2/6, AAV2/8.2 and AAV2/8 (see textfor details). The dose levels ranged from 5e10 to 1e12 viral genomes,and three mice were injected per group. Viral genomes present perdiploid cell were also calculated and are indicated at the bottom ofeach lane. The percent indels induced by each treatment is alsoindicated below each lane and demonstrates that this ZFN pair is capableof cleaving the albumin locus. Control mice were injected with phosphatebuffered saline. A non-specific band is also indicated in the figure.

FIG. 7 is a graph depicting the expression of human factor IX (F.IX)from a transgene inserted into the mouse albumin locus in vivo. A humanF.IX donor transgene was inserted into either the mouse albumin locus atintron 1 or intron 12 following cleavage with mouse albumin-specificZFNs in wild type mice. The graph shows expression levels of F.IX over aperiod of 8 weeks following injection of the vectors. ZFN pairstargeting either intron 1 or intron 12 of mouse albumin were used inthis experiment, as well as ZFNs targeted to a human gene as a control.The donor F.IX gene was designed to be used following insertion intointron 1, and thus is not expressed properly when inserted into intron12. The human F.IX transgene is expressed at a robust level for at least8 weeks following insertion into the mouse albumin intron 1 locus.

FIGS. 8A and 8B are graphs depicting the expression and functionality ofthe human F.IX gene in the plasma of hemophilic mice followingZFN-induced F.IX transgene insertion. The experiment described in FIG. 7was repeated in hemophilic mice using the albumin intron 1 specific ZFNsand the human F.IX donor. Two weeks following treatment, expressionlevel in the serum (FIG. 8A) and clotting time (FIG. 8B) were analyzed.The expression of the human F.IX transgene in hemophilic mice was ableto restore clotting time to that of normal mice.

FIG. 9 (SEQ ID NO:82) provides a segment of the human albumin genesequence encompassing parts of exon 1 and intron 1. Horizontal bars overthe sequence indicate the target sites of the zinc finger nucleases.

FIG. 10 shows an alignment of a segment of the albumin genes in intron 1from a variety of primate species including human, H. sapiens (SEQ IDNO:154), cynologous monkey variants (where sequences ‘C’ and ‘S’ derivefrom two different genome sequence sources): M. fascicularis_c (SEQ IDNO:155) and M. fascicularis_s (SEQ ID NO:156) and rhesus, M mulatta (SEQID NO:157). The figure depicts the DNA target locations of the albuminspecific TALENs (indicated by the horizontal bars above the sequence).

FIGS. 11A through 11C show the results of a Cel-I assay carried out ongenomic DNA isolated from HepG2 cells treated with TALENs or ZFNstargeted to human albumin (FIGS. 11A and B) and NHEJ activity of TALENswith different gap spacings (FIG. 11C). The nucleases were introducedinto HepG2 cells via transient plasmid transfection and quantified 3days later for target modification via the Cel-I assay. Two variationsof the TALE DNA binding domain were used, which differed in the locationof their C-terminal truncation points, the +17 version and the +63version (see text). Pairs used are described in Table 10. In addition,three ZFN pairs were also tested and the % indels detected by the Cel 1assay is indicated at the bottom of the lanes. FIG. 11C is a graphdepicting NHEJ activity in terms of the gap spacing (bp) between TALENbinding sites.

FIGS. 12A through 12C depict the results of ZFN pairs directed to therhesus macaque albumin locus. FIG. 12A shows the percent of NHEJactivity for the 35396/36806 pair in comparison with the 35396/36797pair, tested in RF/6A cells in 3 independent experiments all done usinga ZFN concentration of 400 ng. FIG. 12B depicts a dose titration for thetwo pairs, from 50 ng of each ZFN to 400 ng where the samples wereanalyzed at day 3 following transduction. The lower half of FIG. 12Bdepicts another experiment comparing the two pairs at day 3 or day 10using 400 ng of ZFN. FIG. 12C depicts the results of the SELEX analysis(done at 100 mM salt concentration) of the three ZFNs that were beingcompared where the size of the bar above the middle line shows theresults for that position that were expected (i.e., a single bar with avalue of 1.0 above the line would mean that every base at that positionanalyzed in the SELEX analysis was the expected base), while bars belowthe line indicate the presence of non-expected bases. Bars that aredivided indicate the relative contributions of other bases.

FIGS. 13A and 13B demonstrate the insertion of a huGLa transgene donor(deficient in patients afflicted with Fabry's disease) into the albuminlocus in mice. FIG. 13A shows a Western blot against the huGLa proteinencoded by the transgene, where the arrow indicates the presumedprotein. Comparison of the mouse samples from those mice that receivedboth ZFN and donor (samples 1-1, 1-2 and 1-3) with the samples thateither received only ZFN (4-1, 4-2, 4-3) or those that only received thehuGLa donor (“hu Fabry donor”), samples 5-1 and 5-2 leads toidentification of a band that coincides with the human liver lysatecontrol. FIG. 13B depicts ELISA results using a huGLa specific ELISAkit, where samples were analyzed from mice either 14 or 30 daysfollowing virus introduction (see text for details). Error barsrepresent standard deviations (n=3). The results demonstrate that themice that received both the ZFN and donor had higher amounts of huGLasignal that those that only received ZFN or only received donor.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for modifying anendogenous albumin gene, for example, for expressing a transgene in asecretory tissue. In some embodiments, the transgene is inserted into anendogenous albumin gene to allow for very high expression levels thatare moreover limited to hepatic tissue. The transgene can encode anyprotein or peptide including those providing therapeutic benefit.

Thus, the methods and compositions of the invention can be used toexpress therapeutically beneficial proteins (from a transgene) fromhighly expressed loci in secretory tissues. For example, the transgenecan encode a protein involved in disorders of the blood, for example,clotting disorders, and a variety of other monogenic diseases. In someembodiments, the transgene can be inserted into the endogenous albuminlocus such that expression of the transgene is controlled by the albuminexpressional control elements, resulting in liver-specific expression ofthe transgene encoded protein at high concentrations. Proteins that maybe expressed may include clotting factors such as Factor VII, FactorVIII, Factor IX, Factor X, Factor XI, Factor XIII, vWF and the like,antibodies, proteins relevant to lyososomal storage, insulin, alpha1-antitrypsin, and indeed any peptide or protein that when so expressedprovides benefit.

In addition, any transgene can be introduced into patient derived cells,e.g. patient derived induced pluripotent stem cells (iPSCs) or othertypes of stem cells (embryonic, hematopoietic, neural, or mesenchymal asa non-limiting set) for use in eventual implantation into secretorytissues. The transgene can be introduced into any region of interest inthese cells, including, but not limited to, into an albumin gene or asafe harbor gene. These altered stem cells can be differentiated forexample, into hepatocytes and implanted into the liver. Alternately, thetransgene can be directed to the secretory tissue as desired through theuse of viral or other delivery systems that target specific tissues. Forexample, use of the liver-trophic adenovirus associated virus (AAV)vector AAV8 as a delivery vehicle can result in the integration of thetransgene at the desired locus when specific nucleases are co-deliveredwith the transgene.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. See, e.g.,U.S. Patent Publication No. 20110301073, incorporated by referenceherein in its entirety.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No.20110301073.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988;U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO01/88197; WO 02/099084 and U.S. Publication No. 20110301073.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to re-synthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingeror TALEN proteins can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any value therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 101 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or non-coding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528, 2008/0131962 and2011/0201055, incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP or TALEN as describedherein. Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

“Secretory tissues” are those tissues that secrete products. Examples ofsecretory tissues that are localized to the gastrointestinal tractinclude the cells that line the gut, the pancreas, and the gallbladder.Other secretory tissues include the liver, tissues associated with theeye and mucous membranes such as salivary glands, mammary glands, theprostate gland, the pituitary gland and other members of the endocrinesystem. Additionally, secretory tissues include individual cells of atissue type which are capable of secretion.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP or TALEDNA-binding domain is fused to an activation domain, the ZFP or TALEDNA-binding domain and the activation domain are in operative linkageif, in the fusion polypeptide, the ZFP or TALE DNA-binding domainportion is able to bind its target site and/or its binding site, whilethe activation domain is able to up-regulate gene expression. When afusion polypeptide in which a ZFP or TALE DNA-binding domain is fused toa cleavage domain, the ZFP or TALE DNA-binding domain and the cleavagedomain are in operative linkage if, in the fusion polypeptide, the ZFPor TALE DNA-binding domain portion is able to bind its target siteand/or its binding site, while the cleavage domain is able to cleave DNAin the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

Nucleases

Described herein are compositions, particularly nucleases, which areuseful targeting a gene for the insertion of a transgene, for example,nucleases that are specific for albumin. In certain embodiments, thenuclease is naturally occurring. In other embodiments, the nuclease isnon-naturally occurring, i.e., engineered in the DNA-binding domainand/or cleavage domain. For example, the DNA-binding domain of anaturally-occurring nuclease may be altered to bind to a selected targetsite (e.g., a meganuclease that has been engineered to bind to sitedifferent than the cognate binding site). In other embodiments, thenuclease comprises heterologous DNA-binding and cleavage domains (e.g.,zinc finger nucleases; TAL-effector nucleases; meganuclease DNA-bindingdomains with heterologous cleavage domains).

A. DNA-Binding Domains

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG (SEQ ID NO: 162) family, the GIY-YIG family, the His-Cystbox family and the HNH family. Exemplary homing endonucleases includeI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Theirrecognition sequences are known. See also U.S. Pat. No. 5,420,032; U.S.Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res.25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994)Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J.Mol. Biol. 280:345-353 and the New England Biolabs catalogue.

In certain embodiments, the nuclease comprises an engineered(non-naturally occurring) homing endonuclease (meganuclease). Therecognition sequences of homing endonucleases and meganucleases such asI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. Seealso U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al.(1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin(1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. In addition, the DNA-binding specificity ofhoming endonucleases and meganucleases can be engineered to bindnon-natural target sites. See, for example, Chevalier et al. (2002)Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128. The DNA-binding domains of the homing endonucleases andmeganucleases may be altered in the context of the nuclease as a whole(i.e., such that the nuclease includes the cognate cleavage domain) ormay be fused to a heterologous cleavage domain.

In other embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TALE DNA bindingdomain. See, e.g., U.S. Patent Publication No. 2011/0301073,incorporated by reference in its entirety herein. The plant pathogenicbacteria of the genus Xanthomonas are known to cause many diseases inimportant crop plants. Pathogenicity of Xanthomonas depends on aconserved type III secretion (T3S) system which injects more than 25different effector proteins into the plant cell. Among these injectedproteins are transcription activator-like effectors (TALE) which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay et at (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TALEs is AvrBs3 from Xanthomonas campestgris pv.Vesicatoria (see Bonas et at (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TALEs contain a centralized domain of tandem repeats,each repeat containing approximately 34 amino acids, which are key tothe DNA binding specificity of these proteins. In addition, they containa nuclear localization sequence and an acidic transcriptional activationdomain (for a review see Schornack S, et at (2006) J Plant Physiol163(3): 256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et at (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas.

Thus, in some embodiments, the DNA binding domain that binds to a targetsite in a target locus (e.g., albumin or safe harbor) is an engineereddomain from a TALE similar to those derived from the plant pathogensXanthomonas (see Boch et al, (2009) Science 326: 1509-1512 and Moscouand Bogdanove, (2009) Science 326: 1501) and Ralstonia (see Heuer et at(2007) Applied and Environmental Microbiology 73(13): 4379-4384); U.S.Patent Publication No. 2011/0301073 and U.S. Patent Publication No.20110145940.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein (e.g., a zinc finger protein that binds to a target site in analbumin or safe-harbor gene). Preferably, the zinc finger protein isnon-naturally occurring in that it is engineered to bind to a targetsite of choice. See, for example, See, for example, Beerli et al. (2002)Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal etal. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr.Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261;6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317;7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent PublicationNos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated hereinby reference in their entireties.

An engineered zinc finger binding or TALE domain can have a novelbinding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in theirentireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-finger zinc finger proteins or TALE domains) may belinked together using any suitable linker sequences, including forexample, linkers of 5 or more amino acids in length. See, also, U.S.Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linkersequences 6 or more amino acids in length. The DNA binding proteinsdescribed herein may include any combination of suitable linkers betweenthe individual zinc fingers of the protein. In addition, enhancement ofbinding specificity for zinc finger binding domains has been described,for example, in co-owned WO 02/077227.

Selection of target sites; DNA-binding domains and methods for designand construction of fusion proteins (and polynucleotides encoding same)are known to those of skill in the art and described in detail in U.S.Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523;6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057;WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 andU.S. Publication No. 20110301073.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-finger zinc finger proteins) may be linked togetherusing any suitable linker sequences, including for example, linkers of 5or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The proteins described herein may include anycombination of suitable linkers between the individual zinc fingers ofthe protein.

B. Cleavage Domains

Any suitable cleavage domain can be operatively linked to a DNA-bindingdomain to form a nuclease. For example, ZFP DNA-binding domains havebeen fused to nuclease domains to create ZFNs—a functional entity thatis able to recognize its intended nucleic acid target through itsengineered (ZFP) DNA binding domain and cause the DNA to be cut near theZFP binding site via the nuclease activity. See, e.g., Kim et al. (1996)Proc Nat'l Acad Sci USA 93(3):1156-1160. More recently, ZFNs have beenused for genome modification in a variety of organisms. See, forexample, United States Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060188987; 20060063231; and InternationalPublication WO 07/014275. Likewise, TALE DNA-binding domains have beenfused to nuclease domains to create TALENs. See, e.g., U.S. PublicationNo. 20110301073.

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease. Heterologous cleavage domains can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral {why are we always using thequalifiers “integral” and “integer”—are these really necessary? Theyjust seem restrictive and their use would seem to open us up toworkarounds}. number of nucleotides or nucleotide pairs can intervenebetween two target sites (e.g., from 2 to 50 nucleotide pairs or more).In general, the site of cleavage lies between the target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a DNA binding domain and two Fok Icleavage half-domains can also be used.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987;20080131962 and 20110201055, the disclosures of all of which areincorporated by reference in their entireties herein. Amino acidresidues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496,498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets forinfluencing dimerization of the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication No. 2008/0131962, the disclosure of which isincorporated by reference in its entirety for all purposes.

In certain embodiments, the engineered cleavage half-domain comprisesmutations at positions 486, 499 and 496 (numbered relative to wild-typeFokI), for instance mutations that replace the wild type Gln (Q) residueat position 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See US Patent Publication No. 20110201055).Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474; 20080131962 and 20110201055.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and20090068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter, for example the galactokinasepromoter which is activated (de-repressed) in the presence of raffinoseand/or galactose and repressed in presence of glucose.

Target Sites

As described in detail above, DNA domains can be engineered to bind toany sequence of choice in a locus, for example an albumin or safe-harborgene. An engineered DNA-binding domain can have a novel bindingspecificity, compared to a naturally-occurring DNA-binding domain.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual (e.g., zinc finger) amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of DNA binding domain which bind the particulartriplet or quadruplet sequence. See, for example, co-owned U.S. Pat.Nos. 6,453,242 and 6,534,261, incorporated by reference herein in theirentireties. Rational design of TAL-effector domains can also beperformed. See, e.g., U.S. Patent Publication No. 20110301073.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO00/27878; WO 01/88197 and GB 2,338,237.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Application Publication Nos. 20050064474 and 20060188987,incorporated by reference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-finger zinc finger proteins) may be linked togetherusing any suitable linker sequences, including for example, linkers of 5or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626; 6,903,185; and7,153,949 for exemplary linker sequences 6 or more amino acids inlength. The proteins described herein may include any combination ofsuitable linkers between the individual DNA-binding domains of theprotein. See, also, U.S. Publication No. 20110301073.

Donors

As noted above, insertion of an exogenous sequence (also called a “donorsequence” or “donor”), for example for correction of a mutant gene orfor increased expression of a wild-type gene. It will be readilyapparent that the donor sequence is typically not identical to thegenomic sequence where it is placed. A donor sequence can contain anon-homologous sequence flanked by two regions of homology to allow forefficient HDR at the location of interest. Additionally, donor sequencescan comprise a vector molecule containing sequences that are nothomologous to the region of interest in cellular chromatin. A donormolecule can contain several, discontinuous regions of homology tocellular chromatin. For example, for targeted insertion of sequences notnormally present in a region of interest, said sequences can be presentin a donor nucleic acid molecule and flanked by regions of homology tosequence in the region of interest.

The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. See, e.g., U.S. Patent Publication Nos. 20100047805 and20110207221. If introduced in linear form, the ends of the donorsequence can be protected (e.g., from exonucleolytic degradation) bymethods known to those of skill in the art. For example, one or moredideoxynucleotide residues are added to the 3′ terminus of a linearmolecule and/or self-complementary oligonucleotides are ligated to oneor both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad.Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphoramidates, and O-methyl ribose ordeoxyribose residues.

A polynucleotide can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,donor polynucleotides can be introduced as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

The donor is generally inserted so that its expression is driven by theendogenous promoter at the integration site, namely the promoter thatdrives expression of the albumin gene. However, it will be apparent thatthe donor may comprise a promoter and/or enhancer, for example aconstitutive promoter or an inducible or tissue specific promoter.

The donor molecule may be inserted into an endogenous gene such thatall, some or none of the endogenous gene is expressed. For example, atransgene as described herein may be inserted into an albumin locus suchthat some or none of the endogenous albumin sequences are expressed, forexample as a fusion with the transgene. In other embodiments, thetransgene (e.g., with or without albumin encoding sequences) isintegrated into any endogenous locus, for example a safe-harbor locus.See, e.g., US patent publications 20080299580; 20080159996 and201000218264.

When albumin sequences (endogenous or part of the transgene) areexpressed with the transgene, the albumin sequences may be full-lengthsequences (wild-type or mutant) or partial sequences. Preferably thealbumin sequences are functional. Non-limiting examples of the functionof these full length or partial albumin sequences include increasing theserum half-life of the polypeptide expressed by the transgene (e.g.,therapeutic gene) and/or acting as a carrier.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered in vivo or ex vivo byany suitable means.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692;6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and7,163,824, the disclosures of all of which are incorporated by referenceherein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using vectors containing sequences encoding one or more of thezinc finger or TALEN protein(s). Any vector systems may be usedincluding, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more of the sequences needed for treatment. Thus, when one ormore nucleases and a donor construct are introduced into the cell, thenucleases and/or donor polynucleotide may be carried on the same vectoror on different vectors. When multiple vectors are used, each vector maycomprise a sequence encoding one or multiple nucleases and/or donorconstructs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and ImmunologyDoerfler and Böhm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26(1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet at (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors is described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6, AAV8, AAV 8.2, AAV9, AAV rh10 and pseudotyped AAV such as AAV2/8,AAV2/5 and AAV2/6 can also be used in accordance with the presentinvention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including non-dividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for anti-tumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides described hereininclude non-integrating lentivirus vectors (IDLV). See, for example, Oryet al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.Patent Publication No 2009/054985.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, a donor polynucleotide can be carried by a plasmid, while theone or more nucleases can be carried by a AAV vector. Furthermore, thedifferent vectors can be administered by the same or different routes(intramuscular injection, tail vein injection, other intravenousinjection, intraperitoneal administration and/or intramuscularinjection. The vectors can be delivered simultaneously or in anysequential order.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

Applications

The methods and compositions of the invention can be used in anycircumstance wherein it is desired to supply a transgene encoding one ormore proteins such that the protein(s) is(are) secreted from thetargeted cell. Thus, this technology is of use in a condition where apatient is deficient in some protein due to problems (e.g., problems inexpression level or problems with the protein expressed as sub- ornon-functioning). Particularly useful with this invention is theexpression of transgenes to correct or restore functionality in clottingdisorders. Additionally, A1AT-deficiency disorders such as COPD or liverdamage, or other disorders, conditions or diseases that can be mitigatedby the supply of exogenous proteins by a secretory organ may besuccessfully treated by the methods and compositions of this invention.Lysosomal storage diseases can be treated by the methods andcompositions of the invention, as are metabolic diseases such asdiabetes.

Proteins that are useful therapeutically and that are typicallydelivered by injection or infusion are also useful with the methods andcompositions of the invention. By way of non-limiting examples,production of a C-peptide (e.g. Ersatta™ by Cebix) or insulin for use indiabetic therapy. A further application includes treatment ofEpidermolysis Bullosa via production of collagen VII. Expression ofIGF-1 in secretory tissue as described herein can be used to increaselevels of this protein in patients with liver cirrhosis and lipoproteinlipase deficiency by expression of lipoprotein lipase. Antibodies mayalso be secreted for therapeutic benefit, for example, for the treatmentof cancers, autoimmune and other diseases. Other proteins related toclotting could be produced in secretory tissue, include fibrinogen,prothrombin, tissue factor, Factor V, Factor XI, Factor XII (Hagemanfactor), Factor XIII (fibrin-stabilizing factor), von Willebrand factor,prekallikrein, high molecular weight kininogen (Fitzgerald factor),fibronectin, antithrombin III, heparin cofactor II, protein C, proteinS, protein Z, protein Z-related protease inhibitor, plasminogen, alpha2-antiplasmin, tissue plasminogen activator, urokinase, plasminogenactivator inhibitor-1, and plasminogen activator inhibitor-2.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN)or TALEN. It will be appreciated that this is for purposes ofexemplification only and that other nucleases can be used, for instancehoming endonucleases (meganucleases) with engineered DNA-binding domainsand/or fusions of naturally occurring of engineered homing endonucleases(meganucleases) DNA-binding domains and heterologous cleavage domains.

EXAMPLES Example 1 Design, Construction and Characterization of ZincFinger Protein Nucleases (ZFN) Targeted to the Mouse Albumin Gene

Zinc finger proteins were designed to target cleavage sites withinintrons 1, 12 and 13 of the mouse albumin gene. Corresponding expressionconstructs were assembled and incorporated into plasmids, AAV oradenoviral vectors essentially as described in Urnov et al. (2005)Nature 435(7042):646-651, Perez et at (2008) Nature Biotechnology26(7):808-816, and as described in U.S. Pat. No. 6,534,261. Table 1shows the recognition helices within the DNA binding domain of exemplarymouse albumin specific ZFPs while Table 2 shows the target sites forthese ZFPs. Nucleotides in the target site that are contacted by the ZFPrecognition helices are indicated in uppercase letters; non-contactednucleotides indicated in lowercase.

TABLE 1 Murine albumin-specific zinc finger nucleases helix designsTarget Design SBS # F1 F2 F3 F4 F5 F6 Intron TSGSLTR RSDALST QSATRTKTSGHLSR QSGNLAR NA 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 30724NO: 1) NO: 2) NO: 3) NO: 4) NO: 5) Intron RSDHLSA TKSNRTK DRSNLSRWRSSLRA DSSDRKK NA 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 30725NO: 6) NO: 7) NO: 8) NO: 9) NO: 10) Intron TSGNLTR DRSTRRQ TSGSLTRERGTLAR TSANLSR NA 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 30732NO: 11) NO: 12) NO: 1) NO: 13) NO: 14) Intron DRSALAR RSDHLSE HRSDRTRQSGALAR QSGHLSR NS 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 30733NO: 15) NO: 16) NO: 17) NO: 18) NO: 19) Intron RSDNLST DRSALAR DRSNLSRDGRNLRH RSDNLAR QSNALNR 13 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID 30759 NO: 20) NO: 15) NO: 8) NO: 21) NO: 22) NO: 23) IntronDRSNLSR LKQVLVR QSGNLAR QSTPLFA QSGALAR NA 13 (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID 30761 NO: 8) NO: 24) NO: 5) NO: 25) NO: 18) IntronDRSNLSR DGRNLRH RSDNLAR QSNALNR NA NA 13 (SEQ ID (SEQ ID (SEQ ID (SEQ ID30760 NO: 8) NO: 21) NO: 22) NO: 23) Intron RSDNLSV HSNARKT RSDSLSAQSGNLAR RSDSLSV QSGHLSR 13 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID 30767 NO: 26) NO: 27) NO: 28) NO: 5) NO: 29) NO: 19) IntronRSDNLSE ERANRNS QSANRTK ERGTLAR RSDALTQ NA 13 (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID 30768 NO: 30) NO: 31) NO: 32) NO: 13) NO: 33) IntronTSGSLTR DRSNLSR DGRNLRH ERGTLAR RSDALTQ NA 13 (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID 30769 NO: 1) NO: 8) NO: 21) NO: 13) NO: 33) IntronQSGHLAR RSDHLTQ RSDHLSQ WRSSLVA RSDVLSE RNQHRKT 12 (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID 30872 NO: 34) NO: 35) NO: 36) NO: 37)NO: 38) NO: 39) Intron QSGDLTR RSDALAR QSGDLTR RRDPLIN RSDNLSV IRSTLRD12 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 30873 NO: 40) NO: 41)NO: 40) NO: 42) NO: 26) NO: 43) Intron RSDNLSV YSSTRNS RSDHLSA SYWSRTVQSSDLSR RTDALRG 12 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 30876NO: 26) NO: 44) NO: 6) NO: 45) NO: 46) NO: 47) Intron RSDNLST QKSPLNTTSGNLTR QAENLKS QSSDLSR RTDALRG 12 (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID 30877 NO: 20) NO: 48) NO: 11) NO: 49) NO: 46) NO: 47)Intron RSDNLSV RRAHLNQ TSGNLTR SDTNRFK RSDNLST QSGHLSR 12 (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 30882 NO: 26) NO: 50) NO: 11)NO: 51) NO: 20) NO: 19) Intron DSSDRKK DRSALSR TSSNRKT QSGALAR RSDHLSRNA 12 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 30883 NO: 10) NO: 52)NO: 53) NO: 18) NO: 54)

TABLE 2 Target sites of murine albumin-specific ZFNs Target SBS #Target site Intron 1 30724 ctGAAGGTgGCAATGGTTcctctctgct_ (SEQ ID NO: 55)Intron 1 30725 ttTCCTGTAACGATCGGgaactggcatc_ (SEQ ID NO: 56) Intron 130732 aaGATGCCaGTTCCCGATcgttacagga_ (SEQ ID NO: 57) Intron 1 30733agGGAGTAGCTTAGGTCagtgaagagaa_ (SEQ ID NO: 58) Intron 13 30759acGTAGAGAACAACATCTAGattggtgg_ (SEQ ID NO: 59) Intron 13 30761ctGTAATAGAAACTGACttacgtagctt_ (SEQ ID NO: 60) Intron 13 30760acGTAGAGAACAACatctagattggtgg_ (SEQ ID NO: 59) Intron 13 30767agGGAATGtGAAATGATTCAGatatata_ (SEQ ID NO: 61) Intron 13 30768ccATGGCCTAACAACAGtttatcttctt_ (SEQ ID NO: 62) Intron 13 30769ccATGGCCtAACAACaGTTtatcttctt_ (SEQ ID NO: 62) Intron 12 30872ctTGGCTGTGTAGGAGGGGAgtagcagt_ (SEQ ID NO: 63) Intron 12 30873ttCCTAAGTTGGCAGTGGCAtgcttaat_ (SEQ ID NO: 64) Intron 12 30876ctTTGGCTTTGAGGATTAAGcatgccac_ (SEQ ID NO: 65) Intron 12 30877acTTGGCTcCAAGATTTATAGccttaaa_ (SEQ ID NO: 66) Intron 12 30882caGGAAAGTAAGATAGGAAGgaatgtga_ (SEQ ID NO: 67) Intron 12 30883ctGGGGTAAATGTCTCCttgctcttctt_ (SEQ ID NO: 68)

Example 2 Activity of Murine Albumin-Specific ZFNs

The ZFNs in Table 1 were tested for the ability to cleave theirendogenous target sequences in mouse cells. To accomplish this,constructs expressing the ZFNs in Table 1 were transfected into Neuro2Acells in the pairings indicated in FIG. 1. Cells were then maintained at37° C. for 3 days or subjected to a hypothermic shock (30° C., seeco-owned US Patent Publication No. 20110041195). Genomic DNA was thenisolated from Neuro2A cells using the DNeasy kit (Qiagen) and subjectedto the Cel-I assay (Surveyor™, Transgenomics) as described in Perez etal, (2008) Nat. Biotechnol. 26: 808-816 and Guschin et al, (2010)Methods Mol Biol. 649:247-56), in order to quantify chromosomalmodifications induced by ZFN-cleavage. In this assay, PCR is used toamplify a DNA fragment bearing the ZFN target site, and then theresultant amplicon is digested with the mismatch-specific nuclease Cel-I(Yang et al, (2000) Biochemistry 39, 3533-3541), followed by resolutionof intact and cleaved amplicon on an agarose gel. By quantifying thedegree of amplicon cleavage, one may calculate the fraction of mutatedalleles in amplicon and therefore in the original cellular pool. Inthese experiments, all ZFN pairs were ELD/KKR FokI mutation pairs(described above).

Results from the Cel-I assay are shown in FIG. 1, and demonstrate thatthe ZFNs are capable of inducing cleavage and consequent mutations attheir respective target sites. The “percent indel” value shown beneatheach lane indicates the fraction of ZFN targets that were successfullycleaved and subsequently mutated during cellular repair of the doublestranded break via NHEJ. The data also demonstrate increased activitywhen the transduction procedure incorporates the hypothermic shock.

Example 3 Canine Albumin-Specific ZFNs

A pair of ZFNs targeting the canine albumin locus was constructed foruse in in vivo models. The pair was constructed as described in Example1, and is shown below in Table 3. The target for each ZFN is provided inTable 4.

TABLE 3 Canine albumin-specific zinc finger nucleases helix designsTarget SBS # F1 F2 F3 F4 F5 Intron 33115 QRSNLDS QSSDLSR YHWYLKK RSDDLSVTSSNRTK 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 83) NO: 46)NO: 84) NO: 85) NO: 86) Intron 34077 QSGNLAR QYTHLVA RSDHLST RSDARTTDRSALAR 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 87) NO: 88)NO: 89) NO: 15)

TABLE 4 Target sites of canine albumin-specific ZFNs Target SBS #Target site Intron 1 33115 agTATTCGTTTGCTcCAAaatatttgcc (SEQ ID NO: 90)Intron 1 34077 aaGTCATGTGGAGAGAAacacaaagagt (SEQ ID NO: 91)

The canine specific ZFNs were tested in vitro for activity essentiallyas described in example 2, except that the canine cell line D17 wasused. As shown in FIG. 2, the ZFNs were shown to generate ˜30% indels inthis study.

Example 4 Non-Human Primate Albumin Specific ZFNs

ZFNs targeting the albumin locus in rhesus macaque monkeys (Macacamulatta) were also made. The pairs were constructed as described aboveand are shown below in Table 5. The targets for the ZFNs are shown inTable 6. As shown below, the human (SEQ ID NO:92) and rhesus macaque(SEQ ID NO:93) sequences for the binding site for SBS#35396 (see below,Table 7 and 8) are perfectly conserved. The differences between thehuman and rhesus sequences are boxed.

Thus, for the development of the rhesus albumin specific pair, 35396 waspaired with a series of partners which were designed to replace thehuman 35364 partner in rhesus. These proteins are shown below (Table 5)along with their target sequences (Table 6).

TABLE 5 Rhesus albumin-specific zinc finger nucleases helix designsTarget Rhesus SBS # F1 F2 F3 F4 F5 Intron 36813 QSGNLAR HLGNLKT LKHHLTDDRSNLSR RLDNRTA 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 94)NO: 95) NO: 8) NO: 96) Intron 36808 QSGNLAR LMQNRNQ LKHHLTD DRSNLSRRSDHLTT 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 97) NO: 95)NO: 8) NO: 98) Intron 36820 QRSNLVR LRMNLTK LKHHLTD DRSNLSR RSDHLTT 1(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 99) NO: 100) NO: 95) NO: 8)NO: 98) Intron 36819 QRSNLVR LRMNLTK LKHHLTD DRSNLSR RSDHLTQ 1 (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 99) NO: 100) NO: 95) NO: 8) NO: 35)Intron 36806 QSGNLAR LMQNRNQ LKHHLTD DRSNLSR RSDHLTQ 1 (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 97) NO: 95) NO: 8) NO: 35)

TABLE 6 Target sites of rhesus albumin-specific ZFNs Target SBS #Target site Intron 1 36813 ttAGGGACAGTTATGAAttcaatcttca_(SEQ ID NO: 101) Intron 1 36808 ttAGGGACAGTTATGAAttcaatcttca_(SEQ ID NO: 101) Intron 1 36820 ttAGGGACAGTTATGAAttcaatcttca_(SEQ ID NO: 101) Intron 1 36819 ttAGGGACAGTTATGAAttcaatcttca_(SEQ ID NO: 101) Intron 1 36806 ttAGGGACAGTTATGAAttcaatcttca_(SEQ ID NO: 101)

The rhesus albumin specific ZFNs were tested in pairs to determine thepair with the greatest activity. In each pair, SBS#35396 was tested withthe potential partners shown in Tables 5 and 6 in the rhesus cell lineRF/6A using the methods described above.

The resultant activity, as determined by percent of mismatch detectedusing the Cel-I assay is shown in the body of the matrix (Table 7), anddemonstrate that the ZFNs pairs have activity against the rhesus albuminlocus.

TABLE 7 Activity at the rhesus macaque albumin locus 36813 36808 3682036819 36806 35396 21% 26% 23% 30% 20.5%

Two pairs were examined more extensively, comparing sequence specificityby SELEX analysis and by a titration of each pair for activity in vitro.The results demonstrate that the 35396/36806 pair was the most desirablelead pair (see FIG. 12).

Comparison of the sequence of the human albumin locus with the sequencesof other non-human primates demonstrates that similar pairs may bedeveloped for work in other primates such as cynologous monkeys (see,FIGS. 3A and 3B).

Example 5 In Vivo Cleavage by ZFNs in Mice

To deliver the albumin-specific ZFNs to the liver in vivo, the normalsite of albumin production, we generated a hepatotropic adeno-associatedvirus vector, serotype 8 expressing the albumin-specific ZFNs from aliver-specific enhancer and promoter (Shen et al, ibid and Miao et al,ibid). Adult C57BL/6 mice were subjected to genome editing at thealbumin gene as follows: adult mice were treated by i.v. (intravenous)injection with 1×10¹¹ v.g. (viral genomes)/mouse of either ZFN pair 1(SBS 30724 and SBS 30725), or ZFN pair 2 (SBS 30872 and SBS 30873) andsacrificed seven days later. The region of the albumin gene encompassingthe target site for pair 1 was amplified by PCR for the Cel-I mismatchassay using the following 2 PCR primers:

(SEQ ID NO: 69) Cel1 F1: 5′ CCTGCTCGACCATGCTATACT 3′ (SEQ ID NO: 70)Cel1R1: 5′ CAGGCCTTTGAAATGTTGTTC 3′

The region of the albumin gene encompassing the target site for pair 2was amplified by PCR for the Cel-I assay using these PCR primers:

(SEQ ID NO: 71) mAlb set4F4: 5′ AAGTGCAAAGCCTTTCAGGA 3′ (SEQ ID NO: 72)mAlb set4R4: 5′ GTGTCCTTGTCAGCAGCCTT 3′

As shown in FIG. 4, the ZFNs induce indels in up to 17% of their targetsites in vivo in this study.

The mouse albumin specific ZFNs SBS30724 and SBS30725 which target asequence in intron 1 were also tested in a second study. Genes forexpressing the ZFNs were introduced into an AAV2/8 vector as describedpreviously (Li et at (2011) Nature 475 (7355): 217). To facilitate AAVproduction in the baculovirus system, a baculovirus containing achimeric serotype 8.2 capsid gene was used. Serotype 8.2 capsid differsfrom serotype 8 capsid in that the phopholipase A2 domain in capsidprotein VP1 of AAV8 has been replaced by the comparable domain from theAAV2 capsid creating a chimeric capsid. Production of the ZFN containingvirus particles was done either by preparation using a HEK293 system ora baculovirus system using standard methods in the art (See Li et al,ibid, see e.g. U.S. Pat. No. 6,723,551). The virus particles were thenadministered to normal male mice (n=6) using a single dose of 200microliter of 1.0e11 total vector genomes of either AAV2/8 or AAV2/8.2encoding the mouse albumin-specific ZFN. 14 days post administration ofrAAV vectors, mice were sacrificed, livers harvested and processed forDNA or total proteins using standard methods known in the art. Detectionof AAV vector genome copies was performed by quantitative PCR. Briefly,qPCR primers were made specific to the bGHpA sequences within the AAV asfollows:

(SEQ ID NO: 102) Oligo200 (Forward) 5′-GTTGCCAGCCATCTGTTGTTT-3′(SEQ ID NO: 103) Oligo201 (Reverse) 5′-GACAGTGGGAGTGGCACCTT-3′(SEQ ID NO: 104) Oligo202 (Probe) 5′-CTCCCCCGTGCCTTCCTTGACC-3′

Cleavage activity of the ZFN was measured using a Cel-I assay performedusing a LC-GX apparatus (Perkin Elmer), according to manufacturer'sprotocol. Expression of the ZFNs in vivo was measured using a FLAG-Tagsystem according to standard methods.

As shown in FIG. 5 (for each mouse in the study) the ZFNs wereexpressed, and cleave the target in the mouse liver gene. The % indelsgenerated in each mouse sample is provided at the bottom of each lane.The type of vector and their contents are shown above the lanes.Mismatch repair following ZFN cleavage (indicated % indels) was detectedat nearly 16% in some of the mice.

The mouse specific albumin ZFNs were also tested for in vivo activitywhen delivered via use of a variety of AAV serotypes including AAV2/5,AAV2/6, AAV2/8 and AAV2/8.2. In these AAV vectors, all the ZFN encodingsequence is flanked by the AAV2 ITRs, contain, and then encapsulatedusing capsid proteins from AAV5, 6, or 8, respectively. The 8.2designation is the same as described above. The SBS30724 and SBS30725ZFNs were cloned into the AAV as described previously (Li et al, ibid),and the viral particles were produced either using baculovirus or aHEK293 transient transfection purification as described above. Dosingwas done in normal mice in a volume of 200 μL per mouse via tailinjection, at doses from 5e10 to 1e12 vg per dose. Viral genomes perdiploid mouse genome were analyzed at days 14, and are analyzed at days30 and 60. In addition, ZFN directed cleavage of the albumin locus wasanalyzed by Cel-I assay as described previously at day 14 and isanalyzed at days 30 and 60.

As shown in FIG. 6, cleavage was observed at a level of up to 21%indels. Also included in Figure are the samples from the previous studyas a comparison (far right, “mini-mouse” study-D14 and a background band(“unspecific band”).

Example 6 In Vivo Co-Delivery of a Donor Nucleic Acid and Albumin ZFNs.

Insertion of human Factor IX: ZFNs were used to target integration ofthe gene for the clotting protein Factor IX (F.IX) into the albuminlocus in adult wild-type mice. In these experiments, the mice weretreated by I.V. injection with either 1×10¹¹ v.g./mouse albumin-specificZFN pair 1 targeting intron 1+donor (“mAlb (intron1)”), 1×10¹¹v.g./mouse albumin-specific ZFN pair 2 targeting intron 12+donor(“mAlb(intron12)”) or a ZFN set that targets a human gene plus donor asa control (“Control”). The ZFN pair #1 was 30724/30725, targeting intron1, and ZFN pair 2 was 30872/30873, targeting exon 12. In theseexperiments, the F.IX donor transgene was integrated via end capturefollowing ZFN-induced cleavage. Alternatively, the F.IX transgene wasinserted into a donor vector such that the transgene was flanked by armswith homology to the site of cleavage. In either case, the F.IXtransgene was the “SA—wild-type hF9 exons 2-8” cassette (see co-ownedU.S. patent application 61/392,333).

Transduced mice were then sampled for serum human F.IX levels, whichwere elevated (see FIG. 7, showing stabilized expression of human F.IXfor at least eight weeks following insertion into intron 1). Theexpressed human F.IX is also functional, as evidenced by the reductionin clotting time in hemophilic mice with a human F.IX transgene targetedinto the albumin locus (see FIG. 8). Notably, within two weeks followingtransgene insertion, the clotting time is not significantly differentthan clotting time in a wild type mouse. When the intron 1 specificdonor was inserted into the intron 12 locus, correct splicing to resultin expression of the huF.IX cannot occur. The lack of signal in thissample verifies that the signal from the intron 1 donor being integratedinto the intron 1 site is truly from correct transgene integration, andnot from random integration and expression at another non-specific site.

Insertion of human alpha galactosidase (huGLa): Similar to the insertionof the human F.IX gene, the gene encoding human alpha galatosidase(deficient in patients with Fabry's disease) was inserted into the mousealbumin locus. The ZFN pair 30724/30725 was used as described aboveusing an alpha galactosidase transgene in place of the F.IX transgene.In this experiment, 3 mice were treated with an AAV2/8 virus containingthe ZFN pair at a dose of 3.0e11 viral genomes per mouse and an AAV2/8virus containing the huGLa donor at 1.5e12 viral genomes per mouse.Control animals were given either the ZFN containing virus alone or thehuGLa donor virus alone. Western blots done on liver homogenates showedan increase in alpha galactosidase-specific signal, indicating that thealpha galactosidase gene had been integrated and was being expressed(FIG. 13A). In addition, an ELISA was performed on the liver lysateusing a human alpha galactosidase assay kit (Sino) according tomanufacturer's protocol. The results, shown in FIG. 13B, demonstrated anincrease in signal in the mice that had been treated with both the ZFNsand the huGLa donor.

Example 7 Design of Human Albumin Specific ZFNs.

To design ZFNs with specificity for the human albumin gene, the DNAsequence of human albumin intron 1 was analyzed using previouslydescribed methods to identify target sequences with the best potentialfor ZFN binding. Regions throughout the intron (loci 1-5) were chosenand several ZFNs were designed to target these regions region (forexample, see FIG. 9 which shows the binding sites of ZFNs from loci1-3). In this analysis, five loci were identified to target in thealbumin intron1 (see FIG. 3B). The target and helices are shown inTables 8 and 9.

TABLE 8 Human albumin-specific zinc finger nucleases helix designsTarget Design SBS # F1 F2 F3 F4 F5 F6 Intron QSSDLSR LRHNLRA DQSNLRARPYTLRL QSSDLSR HRSNLNK 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID 35393 NO: 46) NO: 105) NO: 106) NO: 107) NO: 46) NO: 108) IntronQSSDLSR HRSNLNK DQSNLRA RPYTLRL QSSDLSR HRSNLNK 1 (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID 35394 NO: 46) NO: 108) NO: 106) NO: 107)NO: 46) NO: 108) Intron QSSDLSR LKWNLRT DQSNLRA RPYTLRL QSSDLSR HRSNLNK1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35396 NO: 46) NO: 109)NO: 106) NO: 107) NO: 46) NO: 108) Intron QSSDLSR LRHNLRA DQSNLRARPYTLRL QSSDLSR HRSNLNK 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID 35398 NO: 46) NO: 105) NO: 106) NO: 107) NO: 46) NO: 108) IntronQSSDLSR HRSNLNK DQSNLRA RPYTLRL QSSDLSR HRSNLNK 1 (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID 35399 NO: 46) NO: 108) NO: 106) NO: 107)NO: 46) NO: 108) Intron QSSDLSR VVKWNLRA DQSNLRA RPYTLRL QSSDLSR HRSNLNK1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35405 NO: 46) NO: 110)NO: 106) NO: 107) NO: 46) NO: 108) Intron QSGNLAR LMQNRNQ LKQHLNETSGNLTR RRYYLRL N/A 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35361NO: 5) NO: 97 NO: 111) NO: 11) NO: 112) Intron QSGNLAR HLGNLKT LKQHLNETSGNLTR RRDWRRD N/A 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35364NO: 5) NO: 94) NO: 111) NO: 11) NO: 113) Intron QSGNLAR LMQNRNQ LKQHLNETSGNLTR RRDWRRD N/A 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35370NO: 5) NO: 97 NO: 111) NO: 11) NO: 113) Intron QRSNLVR TSSNRKT LKHHLTDTSGNLTR RRDWRRD N/A 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35379NO: 99) NO: 53) NO: 95) NO: 11) NO: 113) Intron DKSYLRP TSGNLTR HRSARKRQSSDLSR VVRSSLKT N/A 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35458NO: 114) NO: 11) NO: 115) NO: 46) NO: 163) Intron TSGNLTR HRSARKRQSGDLTR NRHHLKS N/A N/A 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35480 NO: 11)NO: 115) NO: 40) NO: 163) Intron QSGDLTR QSGNLHV QSAHRKN STAALSY TSGSLSRRSDALAR 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID 35426 NO: 40)NO: 117) NO: 118) NO: 119) NO: 120) NO: 41) Intron QSGDLTR QRSNLNIQSAHRKN STAALSY DRSALSR RSDALAR 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID 35428 NO: 40) NO: 121) NO: 118) NO: 119) NO: 52) NO: 41)

TABLE 9 Target sites of Human albumin-specific ZFNs Target SBS #Target site Intron 1 35393 ccTATCCATTGCACTATGCTttatttaa (locus 2)(SEQ ID NO: 127) Intron 1 35394 ccTATCCATTGCACTATGCTttatttaa (locus 2)(SEQ ID NO: 127) Intron 1 35396 ccTATCCATTGCACTATGCTttatttaa (locus 2)(SEQ ID NO: 127) Intron 1 35398 ccTATCCATTGCACTATGCTttatttaa (locus 2)(SEQ ID NO: 127) Intron 1 35399 ccTATCCATTGCACTATGCTttatttaa (locus 2)(SEQ ID NO: 127) Intron 1 35405 ccTATCCATTGCACTATGCTttatttaa (locus 2)(SEQ ID NO: 127) Intron 1 35361 ttTGGGATAGTTATGAAttcaatcttca (locus 2)(SEQ ID NO: 128) Intron 1 35364 ttTGGGATAGTTATGAAttcaatcttca (locus 2)(SEQ ID NO: 128) Intron 1 35370 ttTGGGATAGTTATGAAttcaatcttca (locus 2)(SEQ ID NO: 128) Intron 1 35379 ttTGGGATAGTTATGAAttcaatcttca (locus 2)(SEQ ID NO: 128) Intron 1 35458 ccTGTGCTGTTGATCTCataaatagaac (locus 3)(SEQ ID NO: 129) Intron 1 35480 ccTGTGCTGTTGATctcataaatagaac (locus 3)(SEQ ID NO: 129) Intron 1 35426 ttGTGGTTTTTAAAtAAAGCAtagtgca (locus 3)(SEQ ID NO: 130) Intron 1 35428 ttGTGGTTTTTAAAtAAAGCAtagtgca (locus 3)(SEQ ID NO: 130) Intron 1 34931 acCAAGAAGACAGActaaaatgaaaata (locus 4)(SEQ ID NO: 131) Intron 1 33940 ctGTTGATAGACACTAAAAGagtattag (locus 4)(SEQ ID NO: 132)

These nucleases were tested in pairs to determine the pair with thehighest activity. The resultant matrices of tested pairs are shown inTables 10 and 11, below where the ZFN used for the right side of thedimer is shown across the top of each matrix, and the ZFN used for theleft side of the dimer is listed on the left side of each matrix. Theresultant activity, as determined by percent of mismatch detected usingthe Cel-I assay is shown in the body of both matrices:

TABLE 10 Activity of Human albumin-specific ZFNs (% mutated targets)35393 35394 35396 35398 35399 35405 ave. 35361 18 19 25 22 23 21 2135364 n.d. 24 23 19 21 21 22 35370 21 19 22 n.d. 22 23 21 35379 21 21n.d. 19 19 21 20

TABLE 11 Activity of Human albumin-specific ZFNs (% mutated targets))35458 35480 ave. 35426 4.5 7 3 35428 4.9 6 3.6(note: ‘n.d.’ means the assay on this pair was not done)

Thus, highly active nucleases have been developed that recognize targetsequences in intron 1 of human albumin.

Example 8 Design of Albumin Specific TALENs

TALENs were designed to target sequences within human albumin intron 1.Base recognition was achieved using the canonical RVD-basecorrespondences (the “TALE code”: NI for A, HD for C, NN for G (NK inhalf repeat), NG for T). TALENs were constructed as previously described(see co-owned U.S. Patent Publication No. 20110301073). Targets for asubset of TALENs were conserved in cynomolgus monkey and rhesus macaquealbumin genes (see FIG. 10). The TALENs were constructed in the “+17”and “+63” TALEN backbones as described in US20110301073. The targets andnumeric identifiers for the TALENs tested are shown below in Table 12.

TABLE 12 Albumin specific TALENs SBS # site # of RVDs SEQ ID NO:  102249gtTGAAGATTGAATTCAta 15 133 102250 gtTGAAGATTGAATTCATAac 17 164 102251gtGCAATGGATAGGTCTtt 15 134 102252 atAGTGCAATGGATAGGtc 15 135 102253atTGAATTCATAACTATcc 15 136 102254 atTGAATTCATAACTATCCca 17 137 102255atAAAGCATAGTGCAATGGat 17 138 102256 atAAAGCATAGTGCAATgg 15 139 102257ctATGCTTTATTTAAAAac 15 140 102258 ctATGCTTTATTTAAAAACca 17 141 102259atTTATGAGATCAACAGCAca 17 142 102260 ctATTTATGAGATCAACAGca 17 158 102261ttCATTTTAGTCTGTCTTCtt 17 143 102262 atTTTAGTCTGTCTTCTtg 15 144 102263ctAATACTCTTTTAGTGTct 16 145 102264 atCTAATACTCTTTTAGTGtc 17 146 102265atAATTGAACATCATCCtg 15 147 102266 atAATTGAACATCATCCTGag 17 148 102267atATTGGGCTCTGATTCCTac 17 149 102268 atATTGGGCTCTGATTCct 15 150 102269ttTTTCTGTAGGAATCAga 15 159 102270 ttTTTCTGTAGGAATCAGag 16 151 102271ttATGCATTTGTTTCAAaa 15 152 102272 atTATGCATTTGTTTCAaa 15 153

The TALENs were then tested in pairs in HepG2 cells for the ability toinduce modifications at their endogenous chromosomal targets, and theresults showed that many proteins bearing the +17 truncation point wereactive. Similarly, many TALENs bearing the +63 truncation point werealso active (see Table 13 and FIG. 11). Note that the pair numbers shownin Table 13 correspond with the pair numbers shown above the lanes inFIG. 11. Side by side comparisons with three sets of non-optimizedalbumin ZFNs showed that the TALENs and ZFNs have activities that are inthe same approximate range.

TABLE 13 TALEN-induced target modification in HepG2-C3a cells % modifi-% modifi- Sample cation, cation, pair TALEN C17 C17 TALEN C63 C63 Gap 1102251:102249 15 102251:102249 0 12 2 102251:102250 0 102251:102250 0 103 102252:102249 0 102252:102249 8.3 15 4 102252:102250 32 102252:1022508.0 13 5 102255:102253 38 102255:102253 21 13 6 102255:102254 43102255:102254 0 11 7 102256:102253 0 102256:102253 23 15 8 102256:10225428 102256:102254 16 13 9 102259:102257 18 102259:102257 15 13 10102259:102258 15 102259:102258 0 11 11 102260:102257 15 102260:102257 1315 12 102260:102258 24 102260:102258 11 13 13 102263:102261 0102263:102261 16 17 14 102263:102262 0 102263:102262 15 16 15102264:102261 0 102264:102261 22 18 16 102264:102262 0 102264:102262 1717 20 102267:102265 47 102267:102265 9.8 13 21 102267:102266 4.7102267:102266 0 11 22 102268:102265 4.2 102268:102265 7.9 15 23102268:102266 10 102268:102266 0 13 24 102271:102269 14 102271:102269 012 25 102271:102270 0 102271:102270 0 11 26 102272:102269 0102272:102269 0 13 27 102272:102270 0 102272:102270 0 12 ZFNs 1735361:35396 31 35361:35396 29 6 18 35426:35458 10 35426:35458 7 6 1934931:33940 7.3 34931:33940 7 6

As noted previously (see co-owned U.S. Patent Publication No.20110301073), the C17 TALENs have greater activity when the gap sizebetween the two TALEN target sites is approximately 11-15 bp, while theC63 TALENs sustain activity at gap sizes up to 18 bp (see FIG. 10, 11Cand Table 13).

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A polynucleotide encoding a non-naturallyoccurring fusion protein comprising (i) a Transcription Activator LikeEffector (TALE) DNA binding domain that binds to an endogenous albumingene, wherein the TALE DNA binding protein comprises a plurality of TALErepeat units, each TALE repeat unit comprising an amino acid RepeatVariable Diresidue (RVD) that binds to a nucleotide in a target sequencein an endogenous albumin gene, wherein the TALE DNA binding domaincomprises a +17 or +63C-terminal truncation and (ii) a cleavage domain,wherein the non-naturally occurring fusion protein cleaves theendogenous albumin gene.
 2. An isolated cell comprising one or morepolynucleotides according to claim
 1. 3. The isolated cell of claim 2,wherein the cell is a stem cell.
 4. The cell of claim 3, wherein thestem cell is selected from the group consisting of an embryonic stemcell (ESC), an induced pluripotent stem cell (iPSC), a hepatic stem celland a liver stem cell.
 5. A kit comprising the polynucleotide accordingto claim
 1. 6. A method of cleaving an endogenous albumin gene in acell, the method comprising: introducing, into the cell, one or morepolynucleotides according to claim 1, under conditions such that the oneor more fusion proteins—are expressed and the endogenous albumin gene iscleaved.
 7. The method of claim 6, wherein the one or morepolynucleotides comprise mRNA.
 8. The method of claim 6, wherein the oneor more polynucleotides are included in one or more expression vectors.9. The method of claim 6, wherein the cell is a liver cell.